Although the potential of gene therapy is considerable, current applications have been limited by the available methods of delivery. No method has yet been identified which is able to produce a safe, targeted and efficient transfer of genetic material. Physical methods of cell delivery such as direct injection or liposome treatment deliver the therapeutic gene to the cell where it can be stably maintained extra-chromosomally for weeks to years depending on the cell type (Hyde et al. Nature 1993, 362, 250-255; Alton et al. Nature Genet. 1993, 5, 135-142). However, extensive monitoring and multiple administrations may be required for long term therapy via this method.
The use of engineered viruses, such as adenoviruses or retroviruses, as delivery and chromosomal integration systems takes advantage of the viral mechanisms to bind to target cells and incorporate DNA into chromosomes, thereby solving the multiple administration problem. These viral based integration systems target chromosomes in a nonspecific to region specific manner (Kotin et al. Proc. Natl. Acad. Sci. USA 1990, 87, 2211-2215). If the insertion occurs in random locations on the chromosome, the result could be the activation of a proto-oncogene or the disruption of a tumor suppressor gene, resulting in cell transformation. In retroviral based delivery vectors, the presence of the viral long terminal repeat (LTR), which has promoter and enhancer activities, can lead to inappropriate expression of downstream genes. This mechanism was studied in the activation of the proto-oncogene c-myc by an avian leukosis virus (ALV) infection (Hayward et al. Nature 1981 290, 475-480).
One possible method to avoid the transformation of a target cell is to design a system that allows for site-specific integration in an accessible but non-essential region of the target cell chromosome. In addition, the integrated DNA should have no enhancer or promoter effects on the surrounding genes.
The transposon Tn7 has the ability to perform site-specific recombination downstream of the bacterial glutamine synthetase (glmS) transcriptional terminator (Craig, N. L., Curr. Topics Microbiol. Immunol. 1996, 204 27-48; Gary et al. J. Mol. Biol. 1996, 257, 301-316). The transposed DNA has no obvious effects on the transcription of glmS or the next downstream gene phoS (Kubo, K. and Craig, N., J. Bacteriol. 1990, 172, 2774-2778; Waddel, C. and Craig, N., Proc. Natl. Acad. Sci. USA 1989, 86, 3958-3962; McKown et al. J. Bacteriol. 1988, 170, 352-358). The glmS gene has significant sequence identity to the human glutamine:fructose amidotransferase 6-phosphate (GFAT) and glutamine-fructose-6-phosphate transaminase (GFPT) genes of yeast, on chromosome XI (Cheret et al. Yeast 1993, 9, 1259-1265), mice (Sayeski et al. Gene 1994, 140, 289-190), and humans (McKnight et al. J. Biol. Chem. 1992, 267, 25208-25212). GFPT is a housekeeping gene that is expressed in all cells, is expressed at higher levels in dividing cells, and is expressed at highest levels in tumor cells, as exemplified by a study conducted in rats (Richards, T. C. and Greengard, O., Biochim. Biophys. Acta 1973, 304, 842-850).
Transposons are mobile genetic elements that have the ability to translocate to a variety of sites on both chromosomal and extra-chromosomal DNA. Although transposons can be divided into subgroups based on their transposition mechanism, they all have similar DNA element structures (Orle, K. and Craig, N., Gene 1991, 104, 125-131). Transposons in their simplest form carry at least two genes. Typically, one gene codes for an antibiotic resistance factor and the second gene encodes one or more transposases. The transposase is an enzyme responsible for the recognition of the transposon DNA element, the insertion site on the target DNA, and for catalyzing the transposition event.
Tn7 is a 14 kb transposon that encodes antibiotic resistance to trimethoprim, streptomycin and spectinomycin (Lichtenstein, C. and Brenner, S., Nature 1982, 297, 601-603). In addition to the antibiotic resistance gene, Tn7 encodes five transposases, tnsA-tnsE, which are responsible for the transposition event (Bainton et al. Cell 1993, 72, 931-943; Orle, K. and Craig, N. 1991, supra). TnsA, B, and C are the core transposase proteins responsible for the transposition event, while TnsD and TnsE provide specific and nonspecific target sequence recognition, respectively (Bainton, et al. 1993, supra).
TnsA is a 31,000 dalton protein whose exact role in transposition is unknown but has been implicated in target strand breakage and transposon insertion (Bainton et al. 1993, supra; Orle, K. and Craig, N. 1991, supra). TnsB is a 81,000 dalton protein that recognizes and binds the Tn7 ends (Arciszewska et al. J. Biol Chem. 1991, 266, 21736-21744; Orle. K. and Craig, N. 1991, supra. TnsC is a 63,000 dalton protein that binds DNA and ATP (Gamas, P. and Craig, N. L., Nuc. Acids Res. 1992, 20, 2525-2532. Although this protein binds ATP, hydrolysis does not appear to be necessary for the transposition event. The hydrolysis of ATP has been implicated in transposition immunity (Arciszewska et al. J. Mol. Biol. 1989, 207, 35-52). TnsD is a 59,000 dalton protein that specifically recognizes and binds the DNA sequence in the attTn7 region (Bainton et al. 1993, supra; Orle, K. and Craig, N. 1991, supra). TnsE is a 61,000 dalton protein that also binds target DNA with little sequence specificity (Bainton et al. 1993, supra; Kubo, K. and Craig, N. J. Bacteriol. 1990, 172, 2774-2778). On the basis of sequence analysis, it appears that the genes tnsA and tnsB are contained within one operon and tnsC-tnsE each contain their own regulatory regions.
Mobile genetic elements also carry additional terminal sequence elements that are required for transposition. The two end elements are 10 to 30 base pairs in length and are either identical or closely related sequences that form a pair of terminal inverted repeats. The end elements play at least two functional roles. They act as a sequence specific binding site for the transposase protein and they signal the end of the transposon DNA sequence. The extreme terminal Tn7 end structures contain 30 bp terminal inverted repeats which are necessary for transposition (Arciszewska et al. J. Mol. Biol. 1989, 207, 35-52).
In addition to the terminal inverted repeat, the Tn7 left end segment (Tn7L) contains three separate, highly related 30 bp binding sites for TnsB (Arciszewska, L. and Craig, N., Nuc. Acids Res. 1991, 19, 5021-5029). The Tn7 right end segment (Tn7R) contains four of these highly related 30 bp TnsB binding sites. This non-symmetric arrangement may be responsible for the directionality of Tn7 insertion. These sites are bound by TnsB in an orderly, but non-cooperative fashion. The Tn7L sites fill from the innermost site towards the left terminal end, while the Tn7R sites fill from the right terminal end.
Although transposons rely on a site-specific DNA binding protein to recognize the end inverted repeats, most elements show little target specificity, resulting in random insertion events in target DNA molecules. Transposon Tn7 is distinguished from other transposons by its ability to insert at high frequency and at a specific location in the E. coil chromosome. The insertion site, known as attTn7, is located between two genes: glmS, which encodes a protein involved in hexosamine biosynthesis, and phoS, which encodes a protein involved in phosphate transport. The insertion of Tn7 at this site occurs in a single orientation in a non-replicative manner, and does not appear to disrupt the function of either glmS or phoS (Kubo, K. and Craig, N., J. Bacteriol. 1990, 172, 2774-2778); Waddel, C. and Craig, N., Proc. Natl. Acad. Sci. USA 1989, 86, 3958-3962; McKnown et al., J. Bacteriol. 1988, 170, 352-358). The recognition-insertion site is characterized by a 30 base sequence that lies in the 3' end of glmS. The recognition sequence is followed by a 25 base spacer region that locates the five base pair insertion point downstream of the glmS transcriptional terminator. The insertion point itself does not appear to have any specific sequence requirements (Kubo, K. and Craig, N. 1990, supra; Waddel, C. and Craig, N. 1989, supra). Although Tn7 preferentially inserts at the attTn7 site, it can also insert at various other sites with little sequence identity with each other and the attTn7 region (Kubo, K. and Craig, N. 1990, supra; Waddel, C. and Craig, N. 1989, supra). The nonspecific mechanism of insertion appears to be dependent on TnsE.
It is now believed that the terminal ends of a Tn7 transposon can be employed as a vector to insert genetic material into a specific site in yeast, plant, animal, or human chromosomes without disrupting the regulation and expression of genes surrounding the insertion site.